title: Light-dependent phosphorylation of Bardet-Biedl syndrome 5 in photoreceptor cells modulates its interaction with arrestin1.
abstract: Arrestins are dynamic proteins that move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the light conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1. Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that Bardet-Biedl syndrome 5 (BBS5) is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions. Our principal findings in this study are threefold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed.
authors.affiliation: Department of Ophthalmology, University of Florida, Box 100284 JHMHC, Gainesville, FL, 32610-0284, USA.
authors.forename: Tyler S
authors.initials: TS
authors.lastname: Smith
authors.forename: Benjamin
authors.initials: B
authors.lastname: Spitzbarth
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Donald R
authors.initials: DR
authors.lastname: Dugger
authors.forename: Gabi
authors.initials: G
authors.lastname: Stern-Schneider
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Sehn
authors.forename: Susan N
authors.initials: SN
authors.lastname: Bolch
authors.forename: J Hugh
authors.initials: JH
authors.lastname: McDowell
authors.forename: Jeremiah
authors.initials: J
authors.lastname: Tipton
authors.forename: Uwe
authors.initials: U
authors.lastname: Wolfrum
authors.forename: W Clay
authors.initials: WC
authors.lastname: Smith
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D054468:Axoneme / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008027:Light; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D028044:Phosphate-Binding Proteins; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D011493:Protein Kinase C / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism* / Q000648:ultrastructure; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019390:Arrestins; C561645:BBS5 protein, mouse; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D028044:Phosphate-Binding Proteins; D010750:Phosphoproteins; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C